The present invention relates to ketoaldonic acids having formed stereogenic centers of R configuration, particularly octulosonic and nonulosonic acids, and methods for synthesizing such sugars using sialic acid aldolase.
A major synthetic value of enzyme catalysis is its usually predictable stereoselectivity. See, e.g., Whitesides et al., Angew. Chem. Int. Ed. Engl., 24:617 (1985); Jones, Tetrahedron, 42:3351 (1986); Yamada et al., Angew. Chem. Int. Ed. Engl., 27:622 (1988); Wong, C-H., Science, 244:1145 (1989); Ohno et al., Org. React., 37:1 (1989); Chen et al., Angew. Chem. Int. Ed. Engl., 28:695 (1989).
A change of stereoselectivity, however, may occur, though very unusual, with different substrate structures, temperatures or solvents. See. e.g., Mohr et al., Helv. Chim. Acta, 66:2501 (1983); Sabbioni et al., J. Chem. Soc. Chem. Commun., 236 (1984); Ohno et al., J. Am. Chem. Soc., 103:2405 (1983); Wang et al., J. Org. Chem., 53:3127 (1988); Lalonde et al., J. Am. Chem. Soc., 103:2405 (1981); Wang et al., J. Org. Chem., 53:2323 (1988); Pham et al., J. Am. Chem. Soc., 111:1935 (1989); Keinan et al., J. Am. Chem. Soc., 108:162 (1986); Sakurai et al., J. Am. Chem. Soc., 110:7236 (1988); Fitzpatrick et al., J. Am. Chem. Soc., 113:3166 (1991). These selectivity changes are often not very significant, with some exceptions where the enantioselectivity is inverted.
In the case of enzymatic aldol reactions, the diastereofacial selectivity for the aldehyde component is often consistent and completely controlled by the enzyme as documented by numerous reactions catalyzed by fructose-1,6-diphosphate (FDP) aldolase or N-acetylneuraminic acid (or sialic acid) aldolase (EC 4.1.3.3). In most cases, the xe2x80x9cDxe2x80x9d isomer of an xcex1-substituted aldehyde reacts faster than the xe2x80x9cLxe2x80x9d isomer, both with si-facial selectivity. The Cram-Felkin mode of attack on the xe2x80x9cDxe2x80x9d aldehyde is therefore proposed for the transition state of the FDP aldolase reaction and the anti-Cram-Felkin mode for the sialic acid aldolase reaction. See. e.g., Toone et al., Tetrahedron, 45:5365 (1989); Bednarski et al., J. Am. Chem. Soc., 111:627 (1989); Straub et al., J. Org. Chem., 55:3926 (1990); Durrwachter et al., J. Org. Chem., 53:4175 (1988); von der Osten et al., J. Am. Chem. Soc., 111:3924 (1989); Kajimoto et al., J. Am. Chem. Soc., 113:6187 (1991); Auge et al., New J. Chem., 12:733 (1988).
Because of the stereoselectivity of enzymes such as aldolases that participate in the metabolism of carbohydrates, it is extremely difficult to design and make new carbohydrates that can be used to study carbohydrate metabolism. There is a need for such synthetic compounds for use as experimental tools in elucidating the molecular character of the numerous and varied pathways involved in carbohydrate anabolism and catabolism.
Of particular relevance to the present invention is the sugar, N-acetylneuraminic acid (NeuAc) or sialic acid. NeuAc is an integral component of most cells and is believed to play a major role in imparting electrical charge characteristics to such cells. Further, NeuAc-like compounds such as the eight and nine-carbon sugar moieties KDO and KDN are major constituents of non-mammalian tissues.
N-Acetylneuraminic Acid (NeuAc) aldolase, also commonly referred to as sialic acid aldolase is a type I aldolase known to form an enamine intermediate with pyruvate, which reversibly reacts with the second substrate N-acetylmannosamine to give NeuAc. See, e.g., Deijl et al., Biochem. Biophys. Res. Commun., 111:668 (1983); and Shukla et al., Anal. Biochem., 158:158 (1986).
NeuAc aldolase is known to accept many aldoses as acceptor substrates. In all previously known aldol condensation reactions with such acceptor substrates, the eneamine intermediate approaches the si face of the incoming aldehyde substrate to form a new stereogenic center of S configuration. Anti-Cram-Felkin attack is generally observed for good chiral aldehyde substrates and Cram-Felkin attack is observed for weak substrates. In both cases, a si-facial selectivity was observed. See. e.g., Auge et al., New J. Chem., 12:733 (1988); and Auge et al., Tetrahedron, 46:201 (1990).
Based on such current knowledge concerning aldolase stereoselectivity, therefore, NeuAc aldolase is considered to be useful only for the production of D-sugars having S configuration. As is disclosed hereinafter, NeuAc aldolase has now unexpectedly been found to be capable of the production of certain ketoaldonic acids having a formed stereogenic center of R configuration.
In one aspect, the present invention contemplates ketoaldonic acids, and particularly octulosonic or nonulosonic acids, having a formed stereogenic center of R configuration. A contemplated ketoaldonic acid is a sialic acid aldolase-catalyzed condensate of pyruvate and an acceptor substrate aldose for that enzyme. The ketoaldonic acid contains a stereogenic center of the R configuration other than present in the acceptor substrate aldose. Exemplary acceptor substrate aldoses include D-gulose and a five or six carbon L-configured acceptor substrate aldose other than L-arabinose, which form an octulosonic or nonulosonic acid. In another aspect, the present invention contemplates a compound having the Formulae I-VIII, below, in which compounds of Formulae V-VIII, are particularly preferred. 
Although L-arabinose forms an octulosonic acid with a new S rather than R stereogenic center in the above sialic acid aldolase-catalyzed condensation with pyruvate, the product of that reaction, 3-deoxy-L-manno-octulosonic acid (L-KDO), a compound of Formula IX, below is new and unexpectedly produced. 
One aspect contemplates a process for preparing a ketoaldonic acid having a new stereogenic center of the R configuration, relative to the aldose starting material. This process comprises the steps of:
(a) admixing in an aqueous solvent (i) pyruvate (typically in excess), (ii) a catalytic amount of sialic acid aldolase and (iii) an acceptor substrate aldose for that enzyme, such as D-gulose or a five or six carbon L-configured acceptor substrate aldose other than L-arabinose, to form a reaction mixture; and
(b) maintaining the reaction mixture for a time period and under biological reaction conditions sufficient for condensation of the pyruvate with the acceptor substrate aldose and the formation of a ketoaldonic acid product.
That product is preferably recovered. Use of D-gulose or a five or six carbon L-configured acceptor substrate aldose forms an octulosonic or nonulosonic acid.
In another process aspect, the present invention contemplates a process for synthesizing a compound of Formulae I-VIII comprising the steps of:
(a) admixing pyruvate (typically in excess), in the presence of a catalytic amount of sialic acid (NeuAc) aldolase, with an acceptor substrate L-rhamnose, L-mannose, L-talose, D-gulose, 2-deoxy-L-glucose, 2-deoxy-L-rhamnose, N-acetyl-L-mannosamine or 2-azido-2-deoxy-L-mannose, respectively, (the latter four aldoses being preferred) to form a reaction mixture; and
(b) maintaining the reaction mixture for a time period and under biological reaction conditions sufficient for condensation of the pyruvate with the acceptor substrate and formation of a compound of Formulae I-VIII, above.
In a preferred embodiment, the synthetic method further comprises recovering the synthesized compound of Formulae I-VIII.
In another embodiment, the invention contemplates an enhanced process for synthesizing any ketoaldonic acid such as an octulosonic or nonulosonic acid like sialic acid. In accordance with this process, an excess of pyruvate, e.g. about 2 to about 10 fold excess, and an acceptor substrate aldose for sialic acid aldolase (EC 4.1.3.3) and a catalytic amount of that aldolase are admixed in an aqueous solvent to form a reaction mixture. That reaction mixture is maintained for a time period and under biological reaction conditions sufficient for the condensation of the pyruvate with the acceptor substrate aldose to form a ketoaldonic acid such as an octulosonic or nonulosonic acid where a five or six carbon acceptor substrate aldose is used.
The enzyme pyruvate decarboxylase is then added and the resulting composition is maintained as above until the excess pryuvate is decomposed. This addition preferably occurs after denaturation of sialic acid aldolase as by acidification. The added pyruvate decarboyxlase can be added as the purified enzyme or as whole acid-free baker""s yeast cells. The ketoaldonic acid is thereafter recovered by standard procedures that include a separation from the yeast cells, where used.
A. Compounds
The present invention contemplates a ketoaldonic acid such as octulosonic and nonulosonic acids. A contemplated ketoaldonic acid is a sialic acid aldolase-catalyzed condensate of pyruvate and an acceptor substrate aldose for that enzyme. The ketoaldonic acid contains a stereogenic center of the R configuration other than present in the acceptor substrate aldose. Exemplary acceptor substrate aldoses include D-gulose and a five or six carbon L-configured acceptor substrate aldose other than L-arabinose, which form an octulosonic or nonulosonic acid. Exemplary octulosonic and nonulosonic acid compounds have the Formulae I, II, III, IV, V, VI, VII or VIII, below, with compounds of Formulae V-VIII being preferred. 
Formula I defines 3,9-dideoxy-L-glycero-L-galactononulosonic acid. Given that 3,9-dideoxy-D-glycero-D-galactononulosonic acid defines D-9-deoxy KDN, the compound of Formula I can also be referred to as L-9-deoxy KDN.
Formula II defines 3-deoxy-L-glycero-L-galactononulosonic acid, which can also be referred to as L-KDN.
The compounds of Formulae I-VIII have a 5C2 conformation as evidenced by the adjacent transaxial coupling of protons at the carbon atoms at positions 3, 4 and 5. Further, the compounds of Formulae I-VIII have a formed stereogenic center of R configuration.
The compounds of Formulae I-VIII synthesized in accordance with the method described herein have a formed stereogenic center of R configuration that is created via the re attack of pyruvate on the acceptor substrate. This re attack and resulting R configuration are surprising and unexpected in view of the published literature. In all previously known aldol condensation reactions using NeuAc aldolase (EC 4.1.3.3), the attack is on the si face of the acceptor substrate and the resulting condensation product has a formed stereogenic center of S configuration. See. e.g., Auge et al., New J. Chem., 12:733 (1988); Auge et al., Tetrahedron, 46:201 (1990); and Kim et al., J. Am. Chem. Soc., 110:6481 (1988).
Thus, where N-acetyl D-mannosamine (D-ManNAc), D-mannose (Man), 4-deoxy-D-Man, 2-deoxy-2-phenyl-D-Man, 6-O-Ac-D-ManNAc, 6-O-Ac-D-Man, 2-deoxy-D-glucose, 6-deoxy-6-N3-D-ManNAc, 6-deoxy-6-F-D-ManNAc, 4,6-dideoxy-4,6-F2-D-talose, D-glucose (D-Glc), D-altrose, 2-deoxy-D-galactose, D-glucosamine (GlcNAc), D-lyxose, D-talose, D-arabinose, L-arabinose or 2-deoxy-ribose was reacted with pyruvate and a catalytic amount of NeuAc aldolase, the resulting condensation products were all found to have formed stereogenic centers of S configuration resulting from a si facial attack. Wong, C-H., Microbial Aldolases in Enzymes in Carbohydrate Synthesis ed. by Bednarski and Simon, American Chemical Society, ACS Symposium Series No. 466 (1991).
The re attack and resulting R configuration where L-rhamnose, L-mannose, L-talose, D-gulose, 2-deoxy-L-glucose, 2-deoxy-L-rhamnose, N-acetyl-L-mannosamine and 2-azido-2-deoxy-L-mannose were used as the acceptor substrate are even more surprising and unexpected because such reversal of stereoselectivity was not observed with all L-isomeric acceptor substrates. Where L-glucose or L-fucose were reacted with pyruvate in the presence of NeuAc aldolase, no aldol condensation product was formed. Wong, C. -H., Microbial Aldolases in Enzymes in Carbohydrate Synthesis ed. by Bednarski and Simon, American Chemical Society, ACS Symposium Series No. 466 (1991).
Another new and useful compound that has a newly formed S rather than R stereogenic center is L-KDO, a compound of Formula IX, below. 
B. Synthetic Process
Another aspect of the present invention contemplates an aldol condensation process of synthesizing a ketoaldonic acid such as an octulosonic or nonulosonic acid that are exemplified by the compounds of Formulae I-VIII. The formed ketoaldonic acid such as an octulosonic or nonulosonic acid contains a new stereogenic center relative to the starting reactants; i.e., not present in the acceptor substrate aldose reactant, and that new stereogenic center has the R configuration. This process comprises the steps of admixing in an aqueous solvent pyruvate (typically in excess), a catalytic amount of sialic acid aldolase and an acceptor substrate aldose for that enzyme such as D-gulose or a five or six carbon L-configured acceptor substrate aldose other than L-arabinose to form a reaction mixture. The reaction mixture is maintained for a time period and under biological reaction conditions sufficient to condense the pyruvate and acceptor substrate aldose and form a desired octulosonic or nonulosonic acid.
In accordance with another aldol condensation process, pyruvate (typically in excess) is admixed in an aqueous solvent in the presence of a catalytic amount of sialic acid (NeuAc) aldolase, with an acceptor substrate as named before to form a reaction mixture. The reaction mixture is maintained for a time period and under biological reaction conditions sufficient to condense the pyruvate and acceptor substrate and form a compound of Formulae I, II, III, IV, V, VI, VII or VIII, as appropriate to the before-noted acceptor substrate, with formation of a compound of Formulae V-VIII being preferred.
The structure of the acceptor substrate dictates the structure of the synthesized aldol condensation product. Where the acceptor substrate is L-rhamnose, the compound of Formula I is synthesized. Where the acceptor substrate is L-mannose, the compound of Formula II is synthesized. Where the acceptor substrate is L-talose, the compound of Formula III is synthesized. Where the acceptor substrate is D-gulose, the compound of Formula IV is synthesized. With 2-deoxy-L-glucose as acceptor substrate, the compound of Formula V is synthesized in a 5:1 ratio to the si face adduct (axial 3-hydroxy group). 2-Deoxy-L-rhamnose is the acceptor substrate for the compound of Formula VI. Where N-acetyl-L-mannosamine is the acceptor substrate, a compound of Formula VII is synthesized. Where 2-azido-2-deoxy-L-mannose is the acceptor substrate, a compound of Formula VIII is formed in a 4.5:1 ratio to the si face adduct (axial 3-hydroxy group).
Pyruvate is readily available from commercial sources (Sigma Chemical Co., St. Louis, Mo.). A preferred formulation of pyruvate is sodium pyruvate. Pyruvate is typically used in an amount in excess of the one mole required for the reaction to drive the reaction to completion. A 2- to about 10-fold excess is usually used.
L-Mannose, L-rhamnose, L-talose and D-gulose are also available from Sigma Chemical Co. 2-Deoxy-L-glucose (Compound 5), 2-deoxy-L-rhamnose (Compound 6), N-acetyl-L-mannosamine (Compound 11) and 2-azido-2-deoxy-L-mannose (Compound 9a) are synthesized as discussed hereinafter.
Highly stable NeuAc aldolase in a free or immobilized form is readily available. See. e.g., Auge et al., New J. Chem., 12:733 (1988); Auge et al., Tetrahedron, 46:201 (1990); and Kim et al., J. Am. Chem. Soc., 110:6481 (1988).
As used herein, the phrase xe2x80x9ccatalytic amountxe2x80x9d means that amount of NeuAc aldolase at least sufficient to catalyze, in a non-rate limiting manner, the condensation of pyruvate and acceptor substrate to product. More than a catalytic amount can be used.
The catalytic amount of NeuAc aldolase varies according to the specific activity of NeuAc aldolase
The reaction time varies with the temperature and the activity of the NeuAc aldolase. Where the NeuAc aldolase has an activity of about 10 Units, the temperature is about 37xc2x0 C., and the concentration of acceptor substrate is about 1 mM, the reaction time is about 48 hours (See Examples 1A and 1B hereafter).
The synthetic method of the present invention can further include recovering a synthesized (formed) ketonaldonic acid such as a compound of Formulae I-VIII. Recovering comprises isolating the synthesized compound from the reaction mixture. Means for isolating a synthesized ketoaldonic acid such as a compound of Formulae I-VIII include gel filtration, column chromatography, paper chromatography, affinity chromatography, extraction, crystallization, precipitation and the like.
In a preferred embodiment, isolation is accomplished by applying a reaction mixture containing about 1 mM acceptor substrate to an anion exchange chromatography column of Dowex(copyright) 1xc3x978xe2x88x92100 (HCOOxe2x88x92 or HCO3xe2x88x92form; 30xc3x972 or 20xc3x972.5 cm) and eluting a compound of Formulae I-VIII with formic acid (0.2M) or bicarbonate (0xe2x86x920.2M), respectively. Product-containing fractions are then pooled, freeze-dried and deionized with Dowex(copyright) W-X8 [H+], and freeze dried again. The pure compounds are finally obtained by bio-gel chromatography. Where such an embodiment is used for isolation, a compound of Formula I can typically be recovered with a yield of about 80 percent (See Example 1A).
The reaction rate of the method of the present invention is within a factor of about 10 of the reaction rate of NeuAc aldolase-catalyzed condensation of pyruvate with acceptor substrates having an enantiomeric configuration (i.e., D-rhamnose, D-mannose, D-talose, L-gulose, 2-deoxy-D-glucose, 2-deoxy-D-(Units/mg), the concentration of acceptor substrate as well as biological reaction conditions such as temperature, time and pH value. Means for determining the catalytic amount of NeuAc aldolase under preselected substrate concentrations and biological reaction conditions are well known to those of skill in the art. Typical amounts range from about 5 to about 20 Units (U) per millimole (mmol) of acceptor substrate, with about 10 to about 15 U/mmol typically being used.
Each ingredient is admixed with each of the other ingredients in a suitable aqueous solvent to form a reaction mixture. The reaction mixture is maintained under biological reaction conditions (temperature, pH, solvent osmolality, ionic composition and ambient pressure) for a period of time sufficient to condense the substrate acceptor and pyruvate to form a compound of Formula I, II, III, IV, V, VI, VII or VIII. A compound of Formula IX can be similarly prepared using L-arabinose as the acceptor substrate aldol.
Temperature can range from about 15xc2x0 C. to about 40xc2x0 C. Preferably, temperature is from about 20xc2x0 C. to about 40xc2x0 C. and, more preferably from about 25xc2x0 C. to about 37xc2x0 C.
The pH value of the solvent and for maintenance can range from about 6.0 to about 11.0. Preferably, the pH value is from about 6.0 to about 8.5 and, more preferably from about 7.0 to about 7.5. The pH value is maintained by buffers in the aqueous solvent. A preferred buffer is potassium phosphate.
The aqueous solvent preferably further comprises an anti-oxidant. A preferred anti-oxidant is a sulfur-containing reducing agent such as a mercaptan (thiol). Exemplary mercaptans are mercaptoethanol and dithiothreitol. rhamnose, N-acetyl-D-mannosamine or 2-azido-2-deoxy-D-mannose). The substantial similarity of the reaction rates with D- and L-configured acceptor substrates is surprising and unexpected. With aldolases other than NeuAc aldolase (i.e., fructose-1,6-diphosphate aldolase), the reaction rate is markedly faster with D-configured acceptor substrates than with L-configured acceptor substrates. See. e.g., Toone et al., Tetrahedron, 45:5365 (1989); Bednarski et al., J. Am. Chem. Soc., 111:627 (1989); Straub et al., J. Org. Chem., 55:3926 (1990); Durrwachter et al., J. Org. Chem., 53:4175 (1988); von der Osten et al., J. Am. Chem. Soc., 111:3924 (1989); Kajimoto et al., J. Am. Chem. Soc., 113:6187 (1991).
An improved process for the synthesis of a ketoaldonic acid such as an octulosonic or nonulosonic acid is contemplated in another embodiment of the invention. This improved process is useful regardless of which enantiomer is prepared; i.e., for any ketoaldonic acid.
Here, an excess of pyruvate, typically about a 2- to about 10-fold excess, is admixed in an aqueous solvent with a catalytic amount of sialic acid aldolase and an acceptor substrate for that enzyme to form a reaction mixture. Specific exemplary D- and L-acceptor substrates are noted before. The reaction mixture is maintained for a time period and under biological reaction conditions sufficient to condense the pyruvate and acceptor substrate to form an octulosonic or nonulosonic acid.
The reaction conditions utilized in this embodiment are as discussed previously. Additional aldose acceptor substrates for sialic acid aldolase are discussed before and are discussed in Wong, C-H., Microbial aldolases in Enzymes in Carbohydrate 
1. 3-Deoxy-D-glycero-L-altro-nonulosonic acid (Compound of Formula III)
[a]25D+32.1xc2x0 (c 0.41, H2O); 1H NMR (D2O, HOD=4.80 ppm) xcex43.98 (1H, ddd, J8-9a=7.0 Hz, J8-9a=5 Hz, J8-7=2.5 Hz, H-8), 3.95 (1H, ddd, J4-3ax=12.5 Hz, J4-5=9.0 Hz, J4-3eq=5.0 Hz, H-4), 3.93 (1H, dd, J7-6=3.5 Hz, J7-8=2.5 Hz, H-7), 3.85 (1H, dd, J6-5=9.5 Hz, J6-73.5 Hz, H-6), 3.69 (1H, dd, J9b-9a=11.5 Hz, J9b-8=5.0 Hz, H-9b), 3.65 (1H, dd, J9a-9b=11.5 Hz, J9a-8=7 Hz, H-9a), 3.56 (1H, t, J5-6=J5-4=9.5 Hz, H-5), 2.21 (1H, dd, J3eq-3ax=12.5 Hz, H-3ax). 13C NMR (D2O+CD3CN) xcex4176.9, 96.7, 74.4, 96.7, 74.4, 72.2, 71.6, 71.1, 69.4, 63.2, 39.3 HRMS (FAB) calcd for C9H16O9Na (M+Na+) 291.0692, found 291.0698.
2. 3,5-Dideoxy-L-glycero-L-galacto-nonulosonic acid (Compound of Formula V)
[a]25D+35.80xc2x0 (c 0.27, H2O); 1H NMR (D2O) xcex44.13 (1H, m, H-4) 4.10 (1H, dt, J6-5ax=12.0 Hz, J6-5eq=J6-7=2.0 Hz, H-6), 3.77 (1H, dd, J9a-9b=12.0 Hz, J9a-8=3.0 Hz, H-9a), 3.72 (1H, ddd, J8-7=9.0 Hz, J8-9a=3.0 Hz, H-8), 3.56 (1H, dd, J9b-9a=12.0 Hz, J9b-8=6.5 Hz, H-9b), 3.41 (1H, dd, J7-8=3.5 Hz, J7-6=1.5 Hz, H-7), 2.03 (1H, ddd, J3eq-3ax=12.5 Hz, J3eq-4=4.5 Hz, J3eq-5eq=1.5 Hz, H-3eq), 1.82 (1H, b dt, J5eq-5ax=12.0 Hz, J5eq-6=J5eq-4=2.0 Hz, H-5eq), 1.56 (1H, dt, J5ax-4=11.5 Hz, J5ax-6=J5ax-5eq=12.0 Hz, H-5ax) 1.49 (1H, t, J3ax-3eq=J3ax-4=12.0 Hz, H-3ax). 13C NMR (D2O+CD3CN) xcex4177.8, 97.4, 73.2, 71.3, 68.6, 64.5, 63.5, 40.3, 35.7. HRMS (FAB) calcd for C9H16O8Na (M+Na+) 275.0743, found 275.0751.
3. 3,5,9-Trideoxy-L-glycero-L-galacto-nonulosonic acid (Compound of Formula VI)
[a]25D+22.1xc2x0 (c 0.19, H2O) 1H NMR (D2O) xcex44.12 (1H, ddt, J4-3eq=5.0 Hz, J4-5eq=2.5 Hz, J4-3ax=J4-5ax=12.0 Hz, H-4), 4.07 (1H, dt, J6-5ax=12.0 Hz, J6-5eq=J6-7=2.5 Hz, H-6), Synthesis, ed. by Bednarski and Simon, American Chemical Society, ACS Symposium Series No. 466 (1991). Non-substrates are also discussed. Whether an aldose is an acceptor substrate for this enzyme can be readily ascertained by admixture of excess pyruvate, the enzyme and potential acceptor substrate aldose as discussed herein, followed by maintenance as discussed herein, e.g. 2-3 days. Analysis of the reaction mixture as by thin layer chromatography indicates whether an octulosonic or nonulosonic acid has been formed.
After the ketoaldonic acid has formed, a catalytic amount of pyruvate decarboxylase is admixed to the aqueous solvent medium and the resulting admixture is maintained as before, but using a pH value of about 5.5 to about 6.5, until the pyruvate has decomposed. This step is utilized because it has been found that the excess pyruvate utilized in the condensation reaction interferes with recovery of the ketoaldonic acid product. Thus, for example, in previously reported procedures for the isolation of enzymatically produced sialic acid [Kim et al., J. Am. Chem. Soc., 110:6481 (1988) and Liu et al., J. Am. Chem. Soc., 114:3901 (1992)] a repetitive extraction of pyruvic acid with ethyl acetate under acidic conditions was used. Under those conditions, the pyruvate exists mainly as the hydrated form in the aqueous phase where its presence makes isolation of sialic acid difficult.
The pyruvate decarboxylase (EC 4.1.1.1) is preferably admixed after denaturation of the sialic acid aldolase. That enzyme is conveniently denatured by adjusting the solution pH value to about 2 and maintaining the pH value for about one hour.
The pyruvate decarboxylase can be provided as a purified enzyme as is available from Sigma Chemical Co. at $80.00 per 100 Units. That enzyme can also be provided by culturing baker""s yeast cells. Baker""s yeast cells are much less costly, e.g. $16.00 per 500 g from Sigma. The baker""s yeast cells must be acid-free, which can be accomplished by washing the cells as described hereinafter.
After the excess pyruvate has been decomposed, the ketoaldonic acid such as octulosonic or nonulosonic acid is recovered by usual techniques such as by ion exchange chromatography or crystallization. Where baker""s yeast cells are used as the source of the pyruvate decarboxylase, the cells are removed as by centrifugation prior to use of ion exchange or other techniques. Exemplary procedures for recovery of the octulosonic or nonulosonic acids are illustrated hereinafter.